Synthesis of oxime nerve agent antidotes

ABSTRACT

The present invention relates to a new route to bis-quaternary pyridinium oximes which can be utilized to restore activity of acetylcholinesterase inhibited by combination with organophosphates.

FIELD

The present invention relates to a synthetic route to bis-quaternarypyridinium oximes which can be utilized to restore activity ofacetylcholinesterase inhibited by combination with organophosphates.

BACKGROUND

Stimulating signals are typically carried by acetylcholine within anervous system synapse. Such signals may be discontinued by a specifictype of cholinesterase enzymes, acetylcholinesterase, which breaks downacetylcholine. If cholinesterase inhibiting chemicals are present, theymay then prevent the breakdown of acetylcholine thereby disruptingnormal nervous system activity. For example, certain chemical classes ofpesticides, such as organophosphates and carbamates, may result in toxiccholinesterase inhibition. Accordingly, if an individual is regularlyexposed to such inhibitors, there remains a need to prophylactically ortherapeutically treat such toxicity. Among other things, individuals oranimals who may have been exposed to a carbamate type cholinesteraseinhibitor may currently be treated with atropine, and those exposed toorganophosphates may beneficially be treated with a pralidoximeantidote.

Organophosphorous nerve agents (OPNA) have been used as chemicalweapons, and as noted, in pesticides, have reportedly cause an estimated300,000 deaths per year worldwide. See, e.g., Eyer, P. et al, Toxicol.Rev. 2003, 22, 165-90. Currently, the bis-pyridinium oximes known as:(1) HLo-7 dimethylsulfate (DMS), otherwise known as1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis[(hydroxyimino)methyl]pyridinium dimethane sulfonate); (2) HI-6 DMS,otherwise known as(1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridiniumdimethane sulfonate); and (3) obidoxime DMS, otherwise known asoxo-[[1-[[4-(oxoazaniumylmethylidene)pyridin-1-yl]methoxymethyl]pyridin-4-ylidene]methyl]azaniumdimethane sulfonate, are reportedly among the most effectivereactivators of OPNA inhibited acetylcholinesterase (AChE).

However, current methods to synthesize the above referenced antidotesrequire the use of chemical compounds which are extremely toxic andwhich lead to relatively large amounts of side products that aredifficult to remove from the reaction media. Accordingly, a continuingneed exists for more efficient pathways to produced desired reactivatorsof OPNA inhibited AChE.

SUMMARY

A method of forming bis-quaternary pyridinium oximes comprising:

supplying benzoic anhydride having the following structure:

reacting said benzoic anhydride with trioxane to form adibenzoyloxymethyl ether having the following structure

supplying a substituted pyridine having the following structure:

wherein R₁ and R₂ may be independently selected from the groupconsisting of hydrogen, alkyl, —CH═NOH or —CONH₂;

combining said dibenzyl acetoxymethyl ether with said substitutedpyridine and forming a salt having the following structure:

reacting said salt with a substituted pyridine having the followingstructure:

wherein R₁ and R₂ may be independently selected from the groupconsisting of hydrogen, alkyl group, —CH═NOH or —CONH₂;

and forming a bis-pyridinium aldoxime salt of the following structure:

wherein R₃ and R₄ may be independently selected from the groupconsisting of consisting of hydrogen, alkyl, —CH═NOH or —CONH₂ and X⁻comprises Cl—, Br—, I— or ⁻OSO₂CH₃.

The present invention also relates to a therapeutic method of treating aperson or animal for intoxication with a phosphorous containingcholinesterase inhibitor, comprising administering to a person or animala bis-pyridinium aldoxime salt formed by the above method.

DETAILED DESCRIPTION

As noted above, current methods to synthesize HLo-7 DMS, HI-6 DMS andobidoxime DMS require use of chemical linkers such asbis(2-chloromethyl) ether (BCME) (2) or bis(2-methylsulfonoxymethyl)ether (BMME) (3), which are identified as extremely carcinogenic, withan exposure limit of 0.0003 ppm. In addition, BMME is relativelydifficult to prepare and is unstable. Below is a summary of the relevantchemical reactions utilized employing such toxic reagents:

In the above scheme, R₁, R₂, R₃ and R₄ may be independently selectedfrom the group consisting of hydrogen, alkyl, —CH═NOH or —CONH₂.Reference to —OMS is reference to —OSO₂CH₃. Reference to X⁻ is thereforereference to the anionic form, either Cl—, Br—, I— or ⁻OSO₂CH₃.Accordingly, in compound 6, when R₁ is hydrogen, R₂ is —CH═NOH, and R₃and R₄ are both —CH═NOH, and X⁻ is ⁻OSO₂CH₃, the compound is HLo-7. WhenR₁=—CH═NOH, R₂ is hydrogen, R3 is hydrogen and R₄ is —CH═NOH, and X⁻ is⁻OSO₂CH₃, the compound is HI-6 DMS. When R₁ and R₃ are hydrogen, and R₂and R₄ are —CH═NOH, and X⁻ is ⁻OSO₂CH₃, the compound is obidoxime DMS.As can be seen from the above, aside from the use of relatively toxicreagents (BCME or BMME), these methods produce relatively large amountsof symmetrical side products 7 and 8, which are relatively difficult toremove from the desired oximes.

The present invention is directed at the formation of adibenzoyloxymethyl ether 10 which can be formed by reacting compound 9,a benzoic anhydride, optionally containing one or more electronwithdrawing groups (EWG) on each of the two aromatic rings, preferablyin the ortho and/or para position with respect to the carbonylfunctionality, with trioxane and H₂SO₄ as an acid catalyst. Preferably,each aromatic ring on compound 9 contains one or two electronwithdrawing groups. Accordingly, the reaction occurs preferably in anorganic solvent, such as dicholorethane (DCE), where the optional andpreferred one or more EWG is illustrated on the reaction scheme below:

In the above, the optional electron withdrawing groups (EWG), ifpresent, may be preferably and independently selected from an organictrihalide (—CF₃, —CCl₃), sulfonate (—SO₃H), nitro group (˜NO₂), ammonium(—NH₃+), aldehyde (—CHO), ketones (—COR), carboxylic acid (—COOH), acylchloride (—COCl), benzoate esters (—COOPh), amide (—CONH₂) or halides(—F, —Cl, —Br, —I). Preferably, the EWG is the same on each of the twoaromatic rings.

The dibenzoyloxymethyl ether 10 can then provide a reactive specieswhich can alkylate substituted pyridines such as compound 1 to form asalt 11:

As illustrated below, when an additional Lewis acid and a secondsubstituted pyridine such as compound 5 is added and reacted withcompound 11 the desired bis-pyridinium OPNA antidotes 6 are formed at ayield in the range of 30-90%. Preferred Lewis acids includetrimethylsilyl iodide (TMSI), Trimethylsilyloxytrifluoromethanesulfonate(TMSOTf), BF₃.OEt₂ or Trimethylsilyloxymethanesulfonate (TMSOMs). Itshould be noted that in this synthetic scheme, R₁, R₂, R₃ and R₄ may beindependently selected from the group consisting of hydrogen, alkyl,—CH═NOH or —CONH₂. In addition, to X⁻ is again reference to the anionicform, either Cl—, Br—, I— or ⁻OSO₂CH₃. Reference to alkyl groupspreferably include, e.g., methyl (—CH₃), ethyl (—CH₂CH₃), propyl(—CH₂CH₂CH₃) or butyl (—CH₂CH₂CH₂CH₃) groups.

Accordingly, the present invention relates to a synthetic procedure forthe formation of bis-quaternary pyridinium oximes which can be utilizedto restore activity of acetylcholinesterase by combination withorganophosphates. The procedure avoids the use of bis(2-chloromethyl)ether (BCME) (2) or bis(2-methylsulfonoxymethyl) ether (BMME) (3) aswell as the formation of compounds 7 and 8 (symmetrical alkylation sideproducts). Moreover, once prepared, the bis-quaternary pyridinium oximesmay be readily incorporated into a pharmaceutically accepted carrier andadministered in an antidotal amount to therapeutically treat exposure toa phosphorous containing cholinesterase inhibitor. A pharmaceuticalaccepted carrier may be understood herein as an aqueous formulationcontaining the bis-quaternary pyridinium oximes in the form of anaqueous solution, suspension or emulsion. The pharmaceuticallyacceptable carrier herein may also include other diluents suitable forpreparing oral pharmaceutical suspensions. For example, pharmaceuticallyacceptable additives including stabilizing agents, suspending agents,surface tension modifiers, viscosity modifiers, colorants,preservatives, flavoring agents.

WORKING EXAMPLES

Unless otherwise noted, solvents and reagents were used withoutpurification. 1,2-dichloroethylene (DCE) was dried over 4 Å molecularsieves for 48 h prior to use. Volatile solvents were removed underreduced pressure using a Buchi rotary evaporator. Infrared (IR) spectrawere obtained using a Nicolet iS550 FT IR spectrophotometer using adiamond crystal attenuated total reflection (ATR) accessory and reportedas wave numbers. Melting points were determined using differentialscanning calorimetry (DSC) on a TA Instruments Differential ScanningCalorimeter Model Q100. Thin layer chromatography (TLC) was performed onglass-backed precoated silica gel plates (0.25 mm thick with 60 F254)and were visualized using one or both of the following manners: UV light(254 nm) and staining with I₂ impregnated silica. Flash chromatographywas performed using the Biotage Isolera One using pre-loaded Silicycle25 g high performance (14-40 μM) columns. ¹H nuclear magnetic resonance(NMR) spectra were obtained at 400 MHz as indicated as solutions inCDCl3 with 0.05% v/v tetramethylsilane (TMS) unless indicated otherwise.¹³C-NMR were obtained at 100 MHz as shown in the indicated deuteratedsolvent. Chemical shifts are reported in parts per million (ppm, δ), andreferenced to TMS, and coupling constants are reported in Hertz (Hz).Spectral splitting patterns are designated as s, singlet; d, doublet; t,triplet; q, quartet; quint, quintuplet; sex, sextet; sept, septuplet; m,multiplet; comp, overlapping multiplets of magnetically nonequivalentprotons; br, broad; and app, apparent.

General Procedure for Anhydride Formation

Pyridine (1 eq) was added to a stirring solution of the benzoic acid (1eq) in CH₂Cl₂ (2 mL/mmol) at room temperature. Thionyl chloride (0.55eq) was added to the solution and was allowed to stir at roomtemperature. The reaction was monitored by TLC (1:1:8CH₂Cl₂/Et₂O/hexanes) and stopped after 2 hours. Diethyl ether (2mL/mmol) was added to the mixture and vortexed. The slurry was filteredthrough a silica plug and washed with ether and the filtrate wasconcentrated under reduced pressure to afford the crude anhydride.

General Procedure for Bis-benzoyloxymethyl Ether Formation

Sulfuric acid (˜0.03 eq) was added to a stirring solution of the benzoicanhydride (1 eq) and trioxane (0.55 eq) in DCE (1.22 mL/mmol). Themixture was heated to 60° C. and monitored by TLC (3:3:4CH₂Cl₂/Et₂O/hexanes) unless otherwise indicated. After 3 hours thereaction was removed from heat and a mixture of 1:1 CH₂Cl₂/H₂O (1.5mL/mmol) was added to the reaction. Ammonium hydroxide (2 drops) wasadded to the mixture and was vortexed. The organic phase was collectedand concentrated under reduced pressure. The residue was preloaded ontosilica and purified by flash chromatography.

bis-benzoyloxymethyl ether (12). Utilized the following TLC conditions:(1:1:8 CH₂Cl₂/Et₂O/hexanes). Isolated 1.31 g (41%) of pure 21a as awhite solid. MP: 50° C. ¹H-NMR (400 MHz) δ 8.02 (app d, J=9.6 Hz, 4H),7.56 (app t, J=7.2 Hz, 2H), 7.39 (t, J=7.6 Hz, 4H), 5.73 (s, 4H);¹³C-NMR (100 MHz) δ 165.8, 133.5, 130.0, 129.4, 128.5, 87.9; IR (pellet)1722 (C═O), 1600, 1452, 1426, 1316, 1264, 1196, 1143, 1113, 1090, 1069,1035, 1025, 1012, 968, 936, 846, 804, 703, 691, 683, 599, 495, 449 cm⁻².

bis-2-chlorobenzoyloxymethyl ether (13). Isolated 477 mg (8%) of pure 13as a clear oil. ¹H-NMR (400 MHz) δ 7.86 (m, 2H), 7.47-7.40 (comp, 4H),7.26 (m, 2H), 5.74 (s, 4H); ¹³C-NMR (100 MHz) δ 164.5, 134.2, 133.1,131.8, 131.2, 128.8, 126.6, 87.9; IR (film) 1731 (C═O), 1591, 1472,1436, 1291, 1243, 1161, 1108, 1072, 1011, 920, 742, 722, 689, 650, 599,473 cm⁻¹.

bis-2-flourobenzoyloxymethyl ether (14). Isolated 162 mg (3%) of pure 14as a clear oil. ¹H-NMR (400 MHz) δ 7.94 (td, J=1.3, 6.1 Hz, 2H), 7.52(m, 2H), 7.16 (t, J=5.8 Hz, 2H), 7.10 (dd, J=7.4, 8.6 Hz, 2H), 5.73 (s,4H); ¹³C-NMR (100 MHz) δ 163.3 (d, J=3.8 Hz), 162.2 (d, J=259.7 Hz),135.1 (d, J=9.1 Hz), 132.3, 124.0 (d, J=4.0 Hz), 117.8 (d, J=9.2 Hz),117.0 (d, J=22.2 Hz), 87.6; IR (film) 1724 (C═O), 1612, 1488, 1456,1293, 1246, 1230, 1154, 1116, 1018, 923, 835, 790, 751, 691, 600, 541,521 cm⁻¹.

bis-2,6-diflourobenzoyloxymethyl ether (15). Isolated 442 mg (7%) ofpure 15 as a white solid. MP: 59° C. ¹H-NMR (400 MHz) δ 7.45 (m, 2H),6.97 (t, J=8.4 Hz, 4H), 5.72 (s, 4H); ¹³C-NMR (100 MHz) δ 161.0 (dd,J=5.8, 256.6 Hz), 160.6, 133.5 (t, J=10.6 Hz), 112.0 (dd, J=3.0, 18.6Hz), 110.1 (t, J=16.9 Hz), 87.1; IR (pellet) 1743 (C═O), 1623, 1465,1284, 1260, 1236, 1150, 1121, 1033, 1008, 962, 923, 884, 829, 799, 772,690, 580, 525, 512, 489, 405 cm⁻¹.

bis-2,6-dichlorobenzoyloxymethyl ether (16). Isolated 264 mg (5%) ofpure 16 as a white solid. MP: 61° C. ¹H-NMR (400 MHz) δ 7.36-7.29 (comp,6H), 5.75 (s, 4H); ¹³C-NMR (500 MHz) δ 163.9, 132.7, 131.9, 131.3,127.9, 87.4; IR (pellet) 1750 (C═O), 1564, 1432, 1289, 1252, 1195, 1158,1126, 1096, 1069, 1012, 965, 945, 911, 814, 800, 774, 751, 724, 605cm⁻¹.

bis-4-chlorobenzoyloxymethyl ether (17). Utilized the following TLCconditions: (2:2:6 CH₂Cl₂/Et₂O/hexanes). Isolated 78 mg (1%) of pure 17as a white solid. MP: 97° C. ¹H-NMR (400 MHz) δ 7.92 (d, J=8.4 Hz, 4H),7.36 (d, J=8.4 Hz, 4H), 5.71 (s, 4H); ¹³C-NMR (100 MHz) δ 164.8, 140.1,131.2, 128.8, 127.6, 88.1; IR (pellet) 1727 (C═O), 1713, 1593, 1487,1457, 1402, 1288, 1267, 1181, 1165, 1090, 1045, 1010, 960, 848, 812,758, 734, 685, 630, 598, 521, 475, 451, cm⁻¹.

bis-4-flourobenzoyloxymethyl ether (18). Utilized the following TLCconditions: (1:1:8 CH₂Cl₂/Et₂O/hexanes). Isolated 698 mg (11%) of pure18 as a white solid. MP: 77° C. ¹H-NMR (400 MHz) δ 8.02 (m, 4H), 7.06(t, J=8.8 Hz, 4H), 5.71 (s, 4H); ¹³C-NMR (100 MHz) δ 166.0 (d, J=253.5Hz), 164.7, 132.4 (d, J=9.4 Hz), 125.5 (d, J=2.9 Hz), 115.6 (d, J=21.9Hz), 88.0; IR (pellet) 1713 (C═O), 1598, 1505, 1460, 1411, 1267, 1234,1217, 1196, 1152, 1110, 1095, 1080, 1031, 1011, 954, 923, 860, 852, 821,792, 763, 688, 585, 542, 499, 485, 465, 423 cm⁻¹.

bis-2,4-dichlorobenzoyloxymethyl ether (19). Utilized the following TLCconditions: (1:1:8 CH₂Cl₂/Et₂O/hexanes). Isolated 3.14 g (28%) of pure19 as a white solid. MP: 80° C. ¹H-NMR (400 MHz) δ 7.82 (d, J=8.4 Hz,2H), 7.46 (d, J=2 Hz, 2H), 7.25 (dd, J=2, 8.4 Hz, 2H)l, 5.51 (s, 4H);¹³C-NMR (100 MHz) δ 163.5, 139.1, 135.5, 132.8, 131.2, 127.0, 126.9,88.2; IR (pellet) 1710 (C═O), 1583, 1469, 1396, 1375, 1292, 1273, 1251,1161, 1130, 1110, 1074, 1024, 972, 933, 895, 841, 826, 781, 762, 675,611, 563, 545, 487, 441. 404 cm⁻¹.

bis-3-chlorobenzoyloxymethyl ether (20). Utilized the following TLCconditions: (1:1:8 CH₂Cl₂/Et₂O/hexanes). Isolated 680 mg (12%) of pure20 as a white solid. MP: 65° C. ¹H-NMR (400 MHz) δ 7.96 (t, J=1.6 Hz,2H), 7.89 (dt, J=1.2, 8.0 Hz, 2H), 7.52 (ddd, J=1.2, 2.0, 8.0 Hz, 2H),7.33 (t, J=7.6 Hz, 2H), 5.72 (s, 4H); ¹³C-NMR (100 MHz) δ 164.5, 134.6,133.5, 130.9, 129.8, 129.7, 127.9, 88.3; IR (pellet) 1713 (C═O), 1574,1469, 1420, 1395, 1295, 1255, 1169, 1118, 1101, 1079, 1059, 1014, 945,897, 890, 809, 749, 737, 672, 658, 575, 497, 464, 424 cm⁻¹.

HI-6 bistriflate (21). Trimethylsilyl trifluoromethanesulfonate (0.92 g,0.75 mL, 4.13 mmol) was added to a 1 dram vial containing linker 10 (250mg, 0.59 mmol), pyridine-2-aldoxime (72 mg, 0.59 mmol),2,6-di-tert-butylpyridine (0.53 g, 0.60 mL, 2.65 mmol), and nitromethane(0.25 mL) and the mixture was stirred at 40° C. for 1.5 h, whereuponisonicotinamide (72 mg, 0.59 mmol) was added and the reactiontemperature raised to 60° C. and stirred for 16 h. The reaction mixturewas then diluted in a mixture (1:1) of MeCN and H₂O (2000 mL) and asample was run on the HPLC according to the HI-6 calibration curvemethod. The area response of the sample was determined to be 32.73 mAU,which corresponds to a 101% yield of HI-6 bistriflate (21) by thefollowing relationship:

${\%\mspace{14mu}{yield}} = \frac{D \times \left( {A + N} \right)}{1000 \times ɛ \times L \times M}$where D is the dilution factor of the sample, A is the absorbance (inmAU), N is the Y intercept, e is the molar absorptivity coefficient, Lis the moles of linker 10 used in the reaction, and M is the molecularweight of HI-6 DMS (478.50 g/mol).

Hlo-7 bistriflate (22). Trifluoromethanesulfonate (0.92 g, 0.75 mL, 4.13mmol) was added to a 1 dram vial containing linker 10 (250 mg, 0.59mmol), pyridine-2,4-dialdoxime (97 mg, 0.59 mmol),2,6-di-tert-butylpyridine (0.53 g, 0.60 mL, 2.65 mmol), and nitromethane(0.25 mL). The mixture was stirred at 40° C. for 1.5 h. Isonicotinamide(72 mg, 0.59 mmol) was added to the mixture and stirred at 60° C. for 16h. The reaction mixture was then diluted in a mixture (1:1) of MeCN andH₂O (800 mL) and a sample was run on the HPLC according to the Hlo-7calibration curve method. The area response of the sample was determinedto be 20.78 mAU, which corresponds to a 29% yield of Hlo-7 bistriflate(22) by the following relationship:

${\%\mspace{14mu}{yield}} = \frac{D \times \left( {A + N} \right)}{1000 \times ɛ \times L \times M}$where D is the dilution factor of the sample, A is the absorbance (inmAU), N is the Y intercept, ε is the molar absorptivity coefficient, Lis the moles of linker 10 used in the reaction, and M is the molecularweight of Hlo-7 DMS (521.52 g/mol).

What is claimed is:
 1. A method of forming bis-quaternary pyridiniumoximes comprising: supplying benzoic anhydride having the followingstructure:

reacting said benzoic anhydride with trioxane to form adibenzoyloxymethyl ether having the following structure:

supplying a substituted pyridine having the following structure:

wherein R₁ and R₂ may be independently selected from the groupconsisting of consisting of hydrogen, alkyl, —CH═NOH or —CONH₂;combining said dibenzoyloxymethyl ether with said substituted pyridineand forming a salt having the following structure:

reacting said salt with a substituted pyridine having the followingstructure:

and forming a bis-pyridinium aldoxime salt of the following structure:

wherein R₃ and R₄ may be independently selected from the groupconsisting of consisting of hydrogen, alkyl, —CH═NOH or —CONH₂ and X⁻comprises Cl— or ⁻OSO₂CH₃.
 2. The method of claim 1 wherein R₁ ishydrogen, R₂ is —CH═NOH, and R₃ and R₄ are both —CH═NOH, and X⁻ is⁻OSO₂CH₃.
 3. The method of claim 1 wherein R₁=—CH═NOH, R₂ is hydrogen,R3 is hydrogen and R₄ is —CH═NOH, and X⁻ is ⁻OSO₂CH₃.
 4. The method ofclaim 1 wherein R₁ and R₃ are hydrogen, and R₂ and R₄ are —CH═NOH, andX⁻ is ⁻OSO₂CH₃.
 5. The method of claim 1 wherein said benzoic anhydridecontains one or more of an electron withdrawing group (EWG) on thearomatic rings according to the following structure:


6. The method of claim 1 wherein said bis-pyridinium aldoxime salt:

is dispersed in a pharmaceutically acceptable carrier.
 7. The method ofclaim 5 wherein said EWG is an organic trihalide.
 8. The method of claim5 wherein said EWG is a sulfonate.
 9. The method of claim 5 wherein saidEWG is a nitro group.
 10. The method of claim 5 wherein said EWG is anammonium group.
 11. The method of claim 5 wherein said EWG is analdehyde.
 12. The method of claim 5 wherein said EWG is a ketone. 13.The method of claim 5 wherein said EWG is a carboxylic acid.
 14. Themethod of claim 5 wherein said EWG is an acyl chloride.
 15. The methodof claim 5 wherein said EWG is a benzoate ester.
 16. The method of claim5 wherein said EWG is an amide group.
 17. The method of claim 5 whereinsaid EWG is a halide.